CN105268022B - A kind of preparation method of xenogenesis cell-less corium ground substance - Google Patents
A kind of preparation method of xenogenesis cell-less corium ground substance Download PDFInfo
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- CN105268022B CN105268022B CN201410377104.4A CN201410377104A CN105268022B CN 105268022 B CN105268022 B CN 105268022B CN 201410377104 A CN201410377104 A CN 201410377104A CN 105268022 B CN105268022 B CN 105268022B
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Abstract
The present invention relates to a kind of preparation method of xenogenesis cell-less corium ground substance, using fresh salted pigskin as raw material, using physics, chemistry and the biochemical method being combined, degreasing, alkali swelling are carried out to pigskin, eliminates a series of processing such as alkali swelling, keratinase processing, secondary enzymatic treatment, a kind of function admirable, widely used xenogenesis (pig) cell-less corium ground substance is prepared.This method is using keratinase processing and secondary enzymatic treatment, this method has the advantages that low in the pollution of the environment and after being handled by this method xenogenesis (pig) cell-less corium ground substance has the advantages that collagen three-dimensional structure is complete, water vapor permeability is good, porosity is high, the good mechanical performance of material, can be widely applied to burn and shaping and beauty field.
Description
Technical field
The present invention relates to bioengineering field, more specifically, is related to one kind and prepares xenogenesis (pig) cell-less corium ground substance
Method
Background technology
Acellular matrix (acellular dermal matrix, ADM) is by skin the methods of utilizing physics, chemistry, biology
Epidermis in skin and the cell containing antigenic component, which thoroughly remove, only to be retained the extracellular matrix of the rack containing collagen in corium and obtains
Arrive, there is the effect such as surface of a wound covering, tissue defect filling, guide tissue regeneration.ADM due to completely without cell component,
Immunocompetence is low, therefore will not generally induce immune response.But ADM remain with normal collagen three-dimensional structure and corium in contain collagen
The extracellular matrix of stent, these components can support the growth of split-thickness skin graft, while be also beneficial to seed cell sticks increasing
Grow, therefore acellular matrix is widely used in burn and shaping and beauty field.Xenogenesis (pig) cell-less corium ground substance is relatively same
Kind of allograft skin has the advantages that to derive from a wealth of sources, is cheap, therefore has huge application prospect.
Pig acellular dermis matrix (porcine acellular dermal matrix, PADM) prepared by conventional method
Removed there are cell be not thorough, poor permeability, the shortcoming such as porosity is low, tensile strength is low.The China of Patent No. CN1387923
Patent discloses a kind of heterogeneous cell-free hypodermal framework and preparation method thereof, the carriage support trypsase and glutaraldehyde processing breast
Pigskin and obtain, the dermis scaffold after the processing of this method has the advantages that tensile strength height, good biocompatibility, but its permeability
Difference, the surface of a wound are also easy to produce hydrops, pneumatosis, and vascularization is slow, and glutaraldehyde is toxic to cell, there is potential danger.Patent No.
The Chinese patent of CN1343523 discloses a kind of the micropore allosome that can be used as wound repair or xenogenesis acellular dermis substitute,
The hole of intensive penetrability is regularly stamped on acellular dermis using the method for machinery or laser, although this can solve nothing
The shortcomings that poor permeability of acellular dermal substitute, but aperture is excessive or overstocked, can increase exposure and the antigen of antigen site
Presenting cells etc. contact the chance of ADM, cause cell compatibility poor, and can destroy the structure of collagen scaffold to a certain extent.
The content of the invention
It is an object of the invention to overcome above-mentioned the deficiencies in the prior art, there is provided a kind of to have good water vapor permeability, resist
The preparation method of the xenogenesis cell-less corium ground substance of Zhang Qiangdu and high porosity.Its main feature is that using salted pigskin as raw material, pass through
The methods of a series of physical, chemistry and biology, prepare the tissue engineering bracket material with medicine utility value.
A kind of preparation method of xenogenesis cell-less corium ground substance is：
(1) weigh：The fresh salted pigskin of correct amount, the meter material foundation as following each operation；
(2) soak：It is being 3.5~4.0 with salted pigskin mass ratio by weighed salted pigskin：1st, temperature 18~25
DEG C, mass concentration be 0.30~0.50% NaCl solution in soak 60~90 minutes, after by pigskin take out control water；
(3) degreasing：It is 3.0~4.0 that material after processing, which is immersed with its mass ratio,:1st, 25~45 DEG C of temperature, quality are dense
Spend the SDS solution for 0.20~0.40%, after immersion 90~120 minutes, then by the material after processing be with its mass ratio
4.0:1 distilled water is washed 2 times, every time 15 minutes；
(4) alkali swelling：It is 3.0~4.0 that material, which is put into its mass ratio,:1st, 18~30 DEG C of temperature, NaOH mass concentrations
In the mixed solution for being 0.3~0.4% for 0.50~0.80% and SDS mass concentrations, in GSD stainless steel temperature control colorimetric rotary drums
It is middle first to rotate 30 minutes, then rotated once at interval of 50 minutes, rotation time is 10 minutes, when total time is 16 small, has been handled
The net waste liquids of Bi Houfang, then by the material after processing with its mass ratio be 4.0:1 distilled water is washed 2 times, every time 15 minutes；
(5) alkali swelling is eliminated：It is being 2.0~3.0 with quality of materials ratio:1st, 25~30 DEG C of temperature, mass concentration for 0.3~
In the solution of 0.6% SDS, the NH that mass concentration is 3.00% is added dropwise4Cl solution, it is 8.0~8.5 to adjust pH value, will
The material that upper step is handled well, which is put into, wherein to react 60~90 minutes, and a pH value was surveyed every 30 minutes, then by after processing
Material with quality of materials ratio be 3.0:1 distilled water is washed 2 times, every time 15 minutes；
(6) keratinase is handled：It is 2.0 that material, which is put into its mass ratio,:1st, 40 DEG C of temperature, pH value 9.5-10.5 it
Between keratinase containing mass concentration 0.20~0.40% and mass concentration 0.2~0.40% SDS mixed solution in it is anti-
Answer 1 it is small when, put net waste liquid after completion of the reaction, then by the material after processing with quality of materials ratio be 2.0:1 distilled water washing 2
It is secondary, 15 minutes every time；
(7) secondary enzymatic treatment：It is being 2.0 with quality of materials ratio:1st, 40 DEG C of temperature, containing 0.30~0.40% pancreas of mass concentration
NH is added in the mixed solution of enzyme and 0.20~0.40%SDS containing mass concentration4Cl solids adjust pH value, its pH value range is existed
Between 7.7-8.5, optimal pH 8；Material is put into the mixed solution after adjusting pH value and is reacted 30 minutes, then it is molten mixing
Liquid add account for 1398 enzymes that concentration of polymer solution is 0.30% react again 2 it is small when, put net waste liquid after completion of the reaction, then will be after processing
Material with quality of materials ratio be 3.0：1 distilled water is washed 3 times, every time 15 minutes；
(8) PBS solution is cleaned：With with quality of materials ratio it is 3.0 at 25 DEG C of temperature：1 PBS solution flushing 3 times, every time
30 minutes.
(9) encapsulate：Material pack sealing；
(10) sterilize：Packaged material cobalt -60100Gy dose irradiations are sterilized, -4 DEG C of gnotobasis is put into and preserves.
Its main technical performance index is as follows：
1 cell-less corium ground substance technical performance index of form
Xenogenesis (pig) cell-less corium ground substance obtained by the method for the present invention has good biocompatibility, excellent
Mechanical property, high porosity, high permeability steam, can be widely applied to burn and shaping and beauty field.
The present invention has the following advantages compared with prior art：
1. environmental pollution is small.Conventional depilating method is in alkaline environment, passes through Na2S/NaHS destroys cystine linkage, makes
Keratin resolves into the peptide or amino acid of small-molecular-weight, so as to achieve the purpose that to remove hair, hair with bits and epidermis.But thisization
Environment can be caused seriously to pollute by learning defeathering technique, lost hair or feathers using keratinase, can thoroughly be abandoned the use of sulfide, be eradicated
Serious pollution of the depilatory solution to environment.
2. collagen three-dimensional structure is complete.Keratinase will not decompose while effectively epidermis, hair is removed with bits and adipose gland
Collagen, can keep the good three-dimensional structure of collagen.
3. the water vapor permeability of material, porosity are high.Experiment shows with the increase of keratinase dosage, the vapor pervious of material
Performance is continuously increased, and when keratinase dosage is in 0.00%-0.30%, the vapor pervious performance of material is slowly increased, and works as dosage
In 0.30%-0.40%, the vapor pervious performance of material significantly increases.When keratinase dosage be more than 0.30 when, material it is saturating
Rate at which moisture is more than 3400gm-224h-1.Meanwhile experiment shows, with the increase of keratinase dosage, the hole of material
Rate is continuously increased, the most obvious to the Porosity Rate Influence of material when keratinase dosage is in 0.00%-0.30%, works as dosage
In 0.30%-0.40%, the porosity increase of material is slow, and enzyme eases up the Porosity Rate Influence of material.When keratinase is used
When amount is more than 0.3%, the vapor pervious speed of material is more than 73%.
4. the good mechanical performance of material.Conventional method can cause the net structure of collagenous fibres to be destroyed, and cause material
Material mechanical strength substantially reduces, the material after being handled with 0.30% keratinase, is ensureing non-collagen tissue removal thoroughly feelings
Under condition, the tensile strength of material is more than 10MPa.
Brief description of the drawings
Fig. 1 is the production technological process of the present invention.
Fig. 2 is xenogenesis (pig) cell-less corium ground substance prepared by the method for the present invention.
Embodiment
The present invention is specifically described below by embodiment
Embodiment 1：
(1) weigh：The salted pigskin of correct amount, the meter material foundation as following each operation；
(2) soak：It is being 3.5 with quality of materials ratio by weighed salted pigskin：1, the mass concentration that 25 DEG C of temperature
To be soaked 60 minutes in 0.50% NaCl solution, after by pigskin take out control water；
(3) degreasing：It is 3.0 that salted pigskin after processing, which is immersed with quality of materials ratio,：The mass concentration that 0 DEG C of Isosorbide-5-Nitrae is
0.40% SDS solution, immersion 90 minutes after, then by the material after processing with quality of materials ratio be 4.0:1 distilled water water
Wash 2 times, every time 15 minutes；
(4) alkali swelling:It is 3.0 that material, which is put into quality of materials ratio, again:1,28 DEG C of temperature, containing mass concentration 0.80%
In the mixed solution of NaOH and the SDS of mass concentration 0.4%, first rotate in rotary drum 30 minutes, then rotated at interval of 50 minutes
Once, rotation time is 10 minutes, total time 16h.Put net waste liquid after being disposed, then the material after processing is used and material
Mass ratio is 4.0:1 distilled water is washed 2 times, every time 15 minutes；
(5) alkali swelling is eliminated:It is being 2.5 with quality of materials ratio:1,30 DEG C of temperature, mass concentration is the solution of 0.6%SDS
In, it is 3.00%NH that mass concentration, which is added dropwise,4Cl solution, it is 8.0~8.5 to adjust pH value, the material that upper step is handled well
It is put into and wherein reacts 90 minutes, and a pH value was surveyed every 30 minutes.Put net waste liquid, then the material after processing is used and material
Mass ratio is 3.0:1 distilled water is washed 2 times, every time 15 minutes；
(6) keratinase is handled:It is 2.0 that material, which is put into quality of materials ratio,:1,40 DEG C of temperature, pH value is in 9.5-10.5
Between keratinase containing mass concentration 0.30% and mass concentration 0.40% SDS mixed solution in reaction 1 it is small when.Instead
Should after put net waste liquid, then by the material after processing with quality of materials ratio be 2.0:1 distilled water washes 2 times, and every time 15
Minute；
(7) secondary enzymatic treatment：It is being 2.0 with quality of materials ratio:1st, 40 DEG C of temperature, the pancreatin containing mass concentration 0.40% and
NH is added in the mixed solution of SDS containing mass concentration 0.40%4Cl solids adjust pH value, make its pH value range in 7.7-8.5
Between, optimal pH 8.Material is put into mixing enzyme solutions and is reacted 30 minutes, then in solution addition and concentration of polymer solution
For 0.30% 1398 enzymes react again 2 it is small when.Put net waste liquid after completion of the reaction, then by the material after processing with quality of materials ratio
For 3.0:1 distilled water is washed 3 times, every time 15 minutes.
(8) PBS liquid cleans：With with quality of materials ratio it is 3.0 at 25 DEG C of temperature：1 PBS solution rinses 3 times, and every time 30
Minute；
(9) encapsulate：Material pack sealing；
(10) sterilize：Packaged material cobalt -60100Gy dose irradiations are sterilized, -4 DEG C of gnotobasis is put into and preserves.
Claims (9)
1. a kind of preparation method of xenogenesis cell-less corium ground substance, it is characterized in that,
（1）Weigh：The fresh salted pigskin of correct amount, the meter material foundation as following each operation；
（2）Immersion：It is being 3.5 ~ 4.0 with quality of materials ratio by weighed salted pigskin:1st, the NaCl of 18 ~ 25 DEG C of temperature is molten
Soaked 60 ~ 90 minutes in liquid, after by pigskin take out control water；
（3）Degreasing：It is 3.0 ~ 4.0 that salted pigskin after processing, which is immersed with quality of materials ratio,:1st, the SDS of 25 ~ 45 DEG C of temperature is molten
Liquid, immersion 90 ~ 120 minutes after, then by the material after processing with quality of materials ratio be 4.0:1 distilled water washing 2 times, often
Secondary 15 minutes；
（4）Alkali swelling：It is 3.0 ~ 4.0 that material, which is put into quality of materials ratio,:1st, the mixing of the NaOH and SDS of 18 ~ 30 DEG C of temperature
In solution, first rotate 30 minutes, then rotated once at interval of 50 minutes, rotation time in GSD stainless steel temperature control colorimetric rotary drums
For 10 minutes, when total time is 16 small, net waste liquid is put after being disposed, then be with quality of materials ratio by the material after processing
4.0:1 distilled water is washed 2 times, every time 15 minutes；
（5）Eliminate alkali swelling：It is being 2.0 ~ 3.0 with quality of materials ratio:1st, in the solution of the SDS of 25 ~ 30 DEG C of temperature, it is added dropwise
Mass concentration is 3.00% NH4Cl solution, and it is 8.0 ~ 8.5 to adjust pH value, and the material that upper step is handled well is put into and is wherein reacted
60 ~ 90 minutes, and every 30 minutes survey a pH value, then by the material after processing with quality of materials ratio be 3.0:1 steaming
Distilled water is washed 2 times, every time 15 minutes；
（6）Keratinase processing：It is 2.0 that material, which is put into quality of materials ratio,:1st, 40 DEG C of temperature, pH values are in 9.5-10.5
Between keratinase and SDS mixed solution in reaction 1 it is small when, put net waste liquid after completion of the reaction, then by after processing
Material is 2.0 with quality of materials ratio:1 distilled water is washed 2 times, every time 15 minutes；
（7）Secondary enzymatic treatment：It is being 2.0 with quality of materials ratio:1st, added in the pancreatin of 40 DEG C of temperature and the mixed solution of SDS
NH4Cl solids adjust pH value, make its pH value range between 7.7-8.5, and material is put into mixed solution and reacts 30 points
Clock, then solution add 1398 enzymes react again 2 it is small when, put net waste liquid after completion of the reaction, then the material after processing is used and material
It is 3.0 to expect mass ratio:1 distilled water is washed 3 times, every time 15 minutes；
（8）PBS solution is cleaned：With with quality of materials ratio it is 3.0 at 25 DEG C of temperature:1 PBS is rinsed 3 times, every time 30 points
Clock；
（9）Encapsulation：Material pack sealing；
（10）Disinfection：Illumination-based disinfection, is put into -4 DEG C of gnotobasis and preserves.
A kind of 2. preparation method of xenogenesis cell-less corium ground substance according to claim 1, it is characterised in that step
（2）, the NaCl concentration of polymer solution is between 0.30 ~ 0.50%.
A kind of 3. preparation method of xenogenesis cell-less corium ground substance according to claim 1, it is characterised in that step（3）
In, the mass concentration of the SDS solution is between 0.20 ~ 0.40%.
A kind of 4. preparation method of xenogenesis cell-less corium ground substance according to claim 1, it is characterised in that step
（4）In, NaOH mass concentrations are between 0.50 ~ 0.80% in NaOH the and SDS mixed solutions, SDS solution qualities
Concentration is between 0.3 ~ 0.4%.
A kind of 5. preparation method of xenogenesis cell-less corium ground substance according to claim 1, it is characterised in that step（5）
In, the mass concentration of the solution of SDS is between 0.3 ~ 0.6%.
A kind of 6. preparation method of xenogenesis cell-less corium ground substance according to claim 1, it is characterised in that step
（6）In, keratinase mass concentration used is between 0.2 ~ 0.40%, between SDS solution concentrations 0.20 ~ 0.40% used.
A kind of 7. preparation method of xenogenesis cell-less corium ground substance according to claim 1, it is characterised in that step
（7）In, the mass concentration of pancreatin used is used between SDS solution concentrations 0.20 ~ 0.40% used between 0.30 ~ 0.40%
The mass concentration of 1398 enzymes is 0.30%.
8. the preparation method of a kind of xenogenesis cell-less corium ground substance according to claim 1, it is characterised in that use
The illumination-based disinfection of cobalt -60, exposure dose 100Gy.
9. xenogenesis cell-less corium ground substance made from claim 1-8 any one of them methods.
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| CN106267312A (en) * | 2016-09-21 | 2017-01-04 | 广东泰宝医疗科技股份有限公司 | A kind of preparation method of xenogenesis high-performance acellular dermal matrix dressing |
| CN107029293B (en) * | 2017-03-03 | 2022-06-21 | 济南金泉生物科技有限公司 | Pericardium collagen membrane for guiding bone regeneration and preparation method and application thereof |
| CN110960729B (en) * | 2019-12-10 | 2021-03-02 | 山东大学 | Polysaccharide modified acellular matrix composite material, preparation and application |
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| CN1526764A (en) * | 2003-09-24 | 2004-09-08 | 山东大学 | Composite collagen-base rack material and its pepn and use |
| CN1569260A (en) * | 2004-05-12 | 2005-01-26 | 四川大学 | Method for preparing acellular dermal matrix material |
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| US8067149B2 (en) * | 1990-09-12 | 2011-11-29 | Lifecell Corporation | Acellular dermal matrix and method of use thereof for grafting |
| AU2003245388A1 (en) * | 2002-06-14 | 2003-12-31 | Crosscart, Inc. | Galactosidase-treated prosthetic devices |
| WO2005063315A1 (en) * | 2003-12-25 | 2005-07-14 | Yoshihiro Takami | Method of preparing isolated cell-free skin, cell-free dermal matrix, method of producing the same and composite cultured skin with the use of the cell-free dermal matrix |
| US20050186286A1 (en) * | 2004-02-25 | 2005-08-25 | Yoshihiro Takami | Skin decellularization method, acellular dermal matrix and production method therefore employing said decellularization method, and composite cultured skin employing said matrix |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1526764A (en) * | 2003-09-24 | 2004-09-08 | 山东大学 | Composite collagen-base rack material and its pepn and use |
| CN1569260A (en) * | 2004-05-12 | 2005-01-26 | 四川大学 | Method for preparing acellular dermal matrix material |
| CN103695514A (en) * | 2013-11-14 | 2014-04-02 | 陕西东大生化科技有限责任公司 | Preparation method for low stimulation polypeptides and oligopeptides, and application of low stimulation polypeptides and oligopeptides in cosmetic field |
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